Our preliminary data indicate that molecularly engineered BOEC (blood outgrowth endothelial cells) can be used for effective gene therapy of hemophilia A. The present project will examine several aspects of BOEC biology that are directly relevant to this potential therapeutic use of BOEC. Three Specific Aims will utilize the model of human BOEC (either unmanipulated or engineered to express human factor VIII) given to NOD. SCID mice. In Aim 1 we will identify the marrow "homing" behavior of BOEC. We will determine where BOEC go in the short- and long-terms when given intravenously. We will identify mechanisms underlying BOEC seeding of marrow and spleen. We will determine if BOEC seeding of marrow/spleen can be increase pharmacologically (with histamine, or beta-1-integrin activating antibody, or VEGF, or 1L6 plus stem cell factor). We will determine if the apparently low seeding frequency of BOEC in marrow is a stochastic or pre-programmed characteristic of establishing a BOEC graft. In Aim 2 we will examine certain issues regarding BOEC graft expansion and longevity in vivo. We will confirm our preliminary impression that BOEC expand substantially in vivo after being administered intravenously. We will determine if this in vivo expansion is predicatble and is dependent on the prior passaging history of the BOEC. We will seek to confirm that silencing of our fVIlI transgene expression vector is not a problem in BOEC. in Aim 3 we expect to document that endothelial expansion in vivo after intravenous administration of BOEC is due to a true BOEC cell rather than a contaminating hematopoietic precursor cell. Each of these studies addresses an issue that is necessary to understand before BOEC technology can be applied for therapeutics. Aside from eventual therapeutics, these studies will define aspects of this unique cell type.